Rotaviruses are the leading cause of severe gastroenteritis in infants and children worldwide. Probiotics, such as Lactobacilli have been shown to reduce the severity of rotavirus diarrhea; however the immunologic mechanisms have not been clearly defined. Colonization of the human intestine with commensal microbes is hypothesized to drive the maturation of the mucosal immune system during neonatal life, but the mechanisms are unknown. Our goal is to define the impact of colonization of the intestine by probiotic commensal microbes on development of the mucosal immune system and innate and adaptive immune responses to enteric virus infections and to clarify the immunological mechanisms involved using gnotobiotic pigs colonized with two actobacillus strains used in the food industry. The key questions that this study will address are: 1) Do commensal microbes enhance maturation of mucosal immunity by triggering toll-like receptors (TLR) and innate immune responses? and 2) Do they act synergistically with viral antigens in the stimulation of TLR expression/activation to induce protective innate and adaptive immunity against rotavirus diarrhea? The specific aims of this study are: (1) To determine the effects of Lactobacilli on intestinal and systemic CD4+ T helper cell (Th) responses (Th 1, Th2 and Th3 cytokine profiles); total (non-specific) and virus-specific effector B cell (antibody-secreting cells); and CD8+ cytotoxic T lymphocyte responses alone and after infection with virulent human rotavirus (HRV). (2) To investigate the effects of Lactobacilli on TLR expressions on blood monocytes/macrophages and dendritic cells and intestinal and systemic innate immune responses (IL-12, IFN-alpha/beta and TNF-alpha cytokine responses) alone and after infection with virulent HRV. An innovative aspect of this study is to determine if non-invasive commensal bacteria like Lactobacilli also trigger TLR (elicit danger signals) like bacterial pathogens. This information is not available, but it is critical to enhance our understanding of the role of TLR in responses to commensal bacteria (probiotics) and the role of commensal bacteria in maturation of mucosal immune system and its innate and acquired components. We will use the unique model of neonatal gnotobiotic pigs which are not colonized by any microbes due to their caesarean derivation and housing in sterile isolator units to investigate the mechanisms and impact of colonization of the gut by a clearly defined Lactobacilli microflora (not confounded by other bacteria) on innate immunity. Further more this model is the only animal model that reproduces the diarrhea seen in infants infected with HRV allowing us to further define the impact of Lactobacilli on HRV disease. Findings from our study will improve our understanding of the development of the mucosal immune system in neonates with or without the influence of the commensal microflora and provide new insights into potential mechanisms for the beneficial effects of probiotics in protective immunity against enteric virus infections. If our results show that Lactobacilli are beneficial, probiotic treatment would likely have the most profound effects in developing countries, where rotavirus diarrhea and mortality are greatest, by providing an inexpensive alternative means to reduce rotavirus diarrhea severity and deaths. The findings from this study will also facilitate an understanding of the potential adjuvant effects of probiotics for development of more effective vaccines against rotavirus and other enteric virus-induced diseases.